The overall goal of this project is to develop a safe and effective new therapeutic agent for influenza virus infections that works by blocking influenza polymerase, which is a unique influenza protein that is critical for virus replication an infection. This project builds on the discovery of new active molecules found by the screening of targeted libraries with innovative influenza polymerase assays. The active molecules that were initially discovered were further developed into new drug-like molecules that are about 100 times more potent in the inhibition of influenza polymerase. The research plan proposes to further develop these compounds into optimized drug leads through additional cycles of library synthesis and directed medicinal chemistry. These lead compounds can then be transferred to GlaxoSmithKline (GSK), who is the product development partner as part of a Partnership for Biodefense. GSK will be responsible for development, clinical trials and distribution of the new first-in-class influenza drug. Some of the major innovations that are associated with this work are; 1) Our original observation regarding the mechanistic similarity between the two-metal binding site of HIV integrase (which is a clinically validated target) and influenza endonuclease, this led to our novel hypothesis that the pharmacophore of these two mechanistically similar sites should share useful common features for targeted library design. 2) The novel specific design and associated development of the high throughput chemistry that allowed for the synthesis of a library of 5,992 new compounds that target the two-metal binding site pharmacophore. 3) Our extension of the internally developed IRdRp mini-genome (replicon) assay that allows for the targeted screening of >10,000 compounds. 4) The design and development of a completely novel in vitro fluorescent polarization (FP) binding assay using a fluorescently labeled known inhibitor of endonuclease and a recombinant PAn endonuclease subunit as the key reagents. 5) Our discovery of novel drug-like compounds that show good cell-based inhibition in the IRdRp replicon assay and also binding in the FP endonuclease assay. 6) Our demonstration that binding in the above FP assay correlates with the published enzyme inhibition for a set of known inhibitors of native influenza endonuclease. 7) The development of a new recombinant PAn construct that has given the first co-crystal structure of an inhibitor with PAn. The Specific Aims of this project are: Aim 1. Lead optimization of novel endonuclease, polymerase and dual inhibitors, using iterative library synthesis and directed medicinal chemistry to select two or more optimized leads for development. Aim 2. Evaluation of the protective and therapeutic efficacy of lead compounds in vitro and in vivo. Aim 3. Conduct lead optimization through non-GLP toxicity and pharmacokinetic studies for the most promising orally-active optimized lead candidates that meet the established criteria.